Antiparasitic macrolide from a strain of Streptomyces hygroscopicus

ABSTRACT

There is disclosed a macrolide isolated from the fermentation broth of a microorganism identified as MA-5285 which morphological analysis reveals to be a strain of Streptomyces hygroscopicus. The compound&#39;s structure is presented based upon analytical studies. The compound has insecticidal and antiparasitic activity.

SUMMARY OF THE INVENTION

This invention is concerned with a novel chemical compound. Inparticular, it is concerned with a novel macrocyclic lactone which isproduced by the fermentation of a nutrient medium with a strain of themicroorganism Streptomyces hygroscopicus MA-5285. Thus, it is an objectof this invention to provide for such novel compound, and a method forpreparing such product microbiologically. It is a further object of thisinvention to provide for the recovery and purification of such compoundfrom the fermentation broth. This substance has antiparasitic andinsecticidal activity, in particular antitapeworm, antigiardiasis andantitrichomonas activity, and it is, thus, an additional object of thisinvention to provide novel antiparasitic and insecticidal compositionscontaining the disclosed compound. Further objects of this inventionwill become apparent from the following description of this invention.

DESCRIPTION OF THE INVENTION

In accordance with this invention, a novel substance is described, whichis prepared by growing under controlled conditions, a previouslyundescribed strain of microorganism. A single novel compound is producedby Streptomyces hygroscopicus MA-5285. The compound is obtained byfermentation and recovered in substantially pure form as describedherein.

Based on taxonomic studies, the microorganism capable of producing thesecompounds are of a new strain of the microorganism Streptomyceshygroscopicus. One such culture, isolted from soil, is designatedMA-5285 in the culture collection of Merck & Co., Inc., Rahway, NewJersey. A sample of this culture, capable of producing the hereindescribed compound, has been deposited, without restriction as toavailability, in the permanent culture collection of the American TypeCulture Collection at 12301 Parklawn Drive, Rockville, Maryland 20852,and has been assigned the accession number ATCC 31955.

The morphological and cultural characteristics of Streptomyceshygroscopicus MA-5285 are set forth below:

CULTURAL CHARACTERISTIC OF:

Streptomyces hygroscopicus MA-5285-ATCC 31955 (V-vegetative growth;A=aerial mycelium; SP=soluble pigment)

Morphology: Sporophores form compact spirals, clustering along aerialmycelia. The coils of spores are hygroscopic, coalescing to form blackmoist droplets. Spore surface is predominantly rugose to warty with somesmooth surfaced spores (transition electron microscopy).

Oatmeal agar (ISP Medium 3)

V: Reverse--tan

A: Dark gray edged with white. As culture ages, the entire gray sporemass becomes black, moist looking and soft.

SP: None

Czapek Dox agar (sucrose nitrate agar)

V: Reverse--pinkish grayed cream

A: Light and dark gray mixed with white

SP: Very light gray

Egg albumin agar

V: Reverse--grayish cream

A: Light gray, moderate. As culture ages, moist black areas appear.

SP: None

Glycerol asparagine agar (ISP Medium 5)

V: Reverse--Dark brown

A: Medium gray edged with white

SP: Light pinkish gray

Inorganic salts-starch agar (ISP Medium 4)

V: Reverse--tan

A: Light gray edged with dark gray. As culture ages, moist black areasappear.

SP: None

Yeast extract-malt extract agar (ISP Medium 2)

V: Reverse--brown

A: Dark gray edged with white. As culture ages, entire gray spore massbecomes black, moist looking and soft.

SP: Light brown

Peptone-iron-yeast extract agar

V: Cream-colored

A: Sparse, whitish

SP: None

Tyrosine agar:

V: Brown edged with dark brown

A: Grayish-white, moderate

SP: Light brown

Carbon Utilization

Pridham-Gottlieb basal medium+1% carbon source; +=growth; ±=growth pooror questionable; -=no growth as compared to negative control (no carbonsource)

    ______________________________________                                                Glucose +                                                                     Arabinose                                                                             ±                                                                  Cellulose                                                                             -                                                                     Fructose                                                                              ±                                                                  Inositol                                                                              ±                                                                  Lactose ±                                                                  Maltose ±                                                                  Mannitol                                                                              ±                                                                  Mannose ±                                                                  Raffinose                                                                             ±                                                                  Rhamnose                                                                              ±                                                                  Sucrose -                                                                     Xylose  ±                                                          ______________________________________                                    

Temperature range (Yeast extract-dextrose+salts agar)

28° C.--Good vegetative growth; good aerial mycelia and sporulation

37° C.--Good vegetative growth; sparse aerial mycelia

50° C.--No growth

Oxygen requirements (Stab culture in yeast extract-dextrose+salts agar)

Aerobic

All readings taken after three weeks at 28° C. unless noted otherwise.pH of all media approximately neutral (6.8-7.2)

A careful comparison of the foregoing data with published descriptions,including Bergey's Manual of Determinative Bacteriology 8th ed (1974);Waksman, The Actinomycetes Vol. II (1961); International Journal ofSystematic Bacteriology 18, 68-189, 279-392 (1968); 19, 391-512 (1969);22, 265-394 (1972); shows a close correlation between the description ofa bacterium identified as Streptomyces hygroscopicus and themorphological and cultural characteristics of MA-5285. It is thereforedetermined that MA-5285 is a strain of the known species Streptomyceshygroscopicus.

The above description is illustrative of a strain of Streptomyceshygroscopicus MA-5285 which can be employed in the production of theinstant compound. However, the present invention also embraces mutantsof the above described microorganism. For example, those mutants whichare obtained by natural selection of those produced by mutating agentsincluding ionizing radiation such as ultraviolet irradiation, orchemical mutagens such as nitrosoguanidine or the like treatments arealso included within the ambit of this invention.

The instant compound is produced during the aerobic fermentation ofsuitable aqueous nutrient media under conditions described hereinafter,with a producing strain of Streptomyces hygroscopicus MA-5285. Aqueousmedia such as those used for the production of many antibioticsubstances are suitable for use in this process for the production ofthis macrocyclic compound.

Such nutrient media contain sources of carbon and nitrogen assimilableby the microorganism and generally low levels of inorganic salts. Inaddition, the fermentation media may contain traces of metals necessaryfor the growth of the microorganisms, and production of the desiredcompound. These are usually present in sufficient concentrations in thecomplex sources of carbon and nitrogen, which may be used as nutrientsources, but can, of course, be added separately to the medium ifdesired.

In general, carbohydrates such as sugars, for example dextrose, sucrose,maltose, lactose, dextran, cerelose, corn meal, oat flour, and the like,and starches are suitable sources of assimilable carbon in the nutrientmedia. The exact quantity of the carbon source which is utilized in themedium will depend, in part, upon the other ingredients in the medium,but it is usually found that an amount of carbohydrate between 0.5 and5% by weight of the medium is satisfactory. These carbon sources may beused individually or several such carbon sources may be combined in thesame medium.

Various nitrogen sources such as yeast hydrolysates, yeast autolysates,yeast cells, tomato paste, corn meal, oat flour, soybean meal, caseinhydrolysates, yeast extracts, corn steep liquors, distillers solubles,cottonseed meal, meat extract and the like, are readily assimilable byStreptomyces hygroscopicus MA-5285 in the production of the instantcompound. The various sources of nitrogen can be used alone or incombination in amounts ranging from 0.2 to 6% by weight of the medium.

Among the nutrient inorganic salts, which can be incorporated in theculture media are the customary salts capable of yielding sodium,potassium, magnesium, ammonium, calcium, phosphate, sulfate, chloride,carbonate, and like ions. Also included are trace metals such as cobalt,manganese, and the like. For the best production of the instantcompound, the addition of calcium carbonate to the production medium ismost preferred.

It should be noted that the media described hereinbelow and in theExamples are merely illustrative of the wide variety of media, which maybe employed, and are not intended to be limitative.

The following are Examples of media suitable for growing strains ofStreptomyces hygroscopicus MA-5285.

    ______________________________________                                        Medium A                                                                      Dextrose                 1.0 g.                                               Soluble starch          10.0 g                                                Beef extract             3.0 g.                                               Yeast autolysate (As ardamine                                                 pH available from Yeast                                                       Products, Inc., Clifton, N.J.)                                                                         5.0 g.                                               NZ Amine-E (caesin hydrolysate-                                               available from Humko-Sheffield-                                               Memphis, Tenn.)          5.0 g.                                               MgSO.sub.4.7H.sub.2 O    0.05 g.                                              Phosphate Buffer         2.0 ml                                               CaCO.sub.3               0.5 g.                                               Distilled water         1000 ml.                                                 pH 7.0-7.2                                                                 Phosphate Buffer                                                              KH.sub.2 PO.sub.4       91.0 g                                                Na.sub.2 HPO.sub.4      95.0 g                                                Distilled water         1000 ml                                                  pH 7.0                                                                     Medium B                                                                      Tomato paste            20.0 g.                                               Primary yeast           10.0 g.                                               Dextrin (CPC starch)    20.0 g.                                               CoCl.sub.2.6H.sub.2 O    0.005 g.                                             Distilled water         1000 ml.                                                 pH 7.2-7.4                                                                 Medium C                                                                      Corn meal               20.0 g.                                               Distillers solubles     10.0 g.                                               Soybean meal            15.0 g.                                               Sodium citrate           4.0 g.                                               CaCl.sub.2.2H.sub.2 O    0.5 g.                                               MgSO.sub.4.7H.sub.2 O    0.1 g.                                               CoCl.sub.2.6H.sub.2 O    0.01 g.                                              FeSO.sub.4.2H.sub.2 O    0.01 g.                                              Polyglycol P2000 (Polypropylene glycol                                        mw 2000)                 2.5 mg.                                              Distilled water         1000 ml.                                                  pH 6.5                                                                    Medium D                                                                      Lactose                 20.0 g.                                               Distillers solubles     15.0 g.                                               Autolyzed yeast (Ardamine pH)                                                                          5.0 g.                                               Distilled water         q.s. to 1000 ml.                                         pH 7.0                                                                     Medium E                                                                      Tomato paste            40.0 g.                                               Oat flour               10.0 g.                                               Distilled water         1000 ml.                                                 pH 7.0                                                                     Medium F                                                                      Corn Steep Liquor       15.0 g.                                               (NH.sub.4).sub.2 SO.sub.4                                                                              4.0 g.                                               CaCO.sub.3               6.0 g.                                               Soluble Starch          20.0 g.                                               Corn meal                1.0 g.                                               Soybean meal             4.0 g.                                               Glucose                  5.0 g.                                               KH.sub.2 PO.sub.4        0.3 g.                                               Lard oil                 2.5 g.                                               Distilled water         1000 ml.                                                 pH 6.7                                                                     Medium G                                                                      Dextrose                10.0 g                                                Asparagine               1.0 g                                                K.sub.2 HPO.sub.4        0.1 g                                                MgSO.sub.4.7H.sub.2 O    0.5 g                                                Yeast Extract            0.5 g                                                Oat Flour               10.0 g                                                CaCO.sub.3               3.0 g                                                Trace Element Mix       10.0 ml                                               Distilled water         1000 ml                                                Adjust pH to 7.2                                                             Trace Element Mix                                                             FeSO.sub.4.7H.sub.2 O   1000 mg                                               MnSO.sub.4.4H.sub.2 O   1000 mg                                               CuCl.sub.2.2H.sub.2 O    25 mg                                                CaCl.sub.2.2H.sub.2 O    100 mg                                               H.sub.3 BO.sub.3         56 mg                                                (NH.sub.4).sub.6 MO.sub.4 O.sub.24.6H.sub.2 O                                                          19 mg                                                ZnSO.sub.4.7H.sub.2 O    200 mg                                               Distilled water         1000 ml                                               Medium H                                                                      Medium G                1000 ml                                               Oat Flour                10 g                                                    pH 7.2                                                                     ______________________________________                                    

The fermentation employing Streptomyces hygroscopicus MA-5285 can beconducted at temperatures ranging from about 20° C. to about 40° C. Foroptimum results, it is most convenient to conduct these fermentations ata temperature in the range of from about 24° C. to about 30° C.Temperatures of about 27°-28° C. are most preferred. The pH of thenutrient medium suitable for producing the instant compounds can varyfrom about 5.0 to 8.5 with a preferred range of from about 6.0 to 7.5.

Small scale fermentations are conveniently carried out by placingsuitable quantities of nutrient medium in a flask employing knownsterile techniques, inoculating the flask with either spores orvegetative cellular growth of Streptomyces hygroscopicus MA-5285 looselystoppering the flask with cotton and permitting the fermentation toproceed in a constant room temperature of about 28° C. on a rotaryshaker at from 95 to 300 rpm for about 2 to 10 days. For larger scalework, it is preferable to conduct the fermentation in suitable tanksprovided with an agitator and a means of aerating the fermentationmedium. The nutrient medium is made up in the tank and aftersterilization is inoculated with a source of vegetative cellular growthof Streptomyces hygroscopicus MA-5285. The fermentation is allowed tocontinue for from 1 to 8 days while agitating and/or aerating thenutrient medium at a temperature in the range of from about 24° to 37°C. The degree of aeration is dependent upon several factors such as thesize of the fermentor, agitation speed, and the like. Generally thelarger scale fermentations are agitated at about 95 to 300 RPM and about2 to 20 cubic feet per minute (CFM) of air.

The novel compound of this invention is found primarily in the myceliumon termination of the Streptomyces hygroscopicus MA-5285 fermentationand may be removed and separated therefrom as described below.

The separation of the novel compound from the whole fermentation brothand the recovery of said compounds is carried out by solvent extractionand application of chromatographic fractionations with variouschromatographic techniques and solvent systems.

The instant compound has slight solubility in water, but is soluble inorganic solvents. This property may be conveniently employed to recoverthe compound from the fermentation broth. Thus, in one recovery method,the whole fermentation broth is combined with approximately an equalvolume of an organic solvent. While any organic solvent may be employed,it is preferable to use a water immiscible solvent such as ethylacetate, methylene chloride, chloroform and the like. Generally severalextractions are desirable to achieve maximum recovery. The solventremoves the instant compound as well as other substances lacking theantiparasitic activity of the instant compound. If the solvent is awater immiscible one, the layers are separated and the organic solventis evaporated under reduced pressure. The residue is placed onto achromatography column containing preferably, silica gel. The columnretains the desired products and some impurities, but lets many of theimpurities, particularly the non-polar impurities, pass through. Thecolumn is washed with a moderately polar organic solvent such asmethylene chloride or chloroform to further remove impurities, and isthen washed with a mixture of methylene chloride or chloroform and anorganic solvent of which acetone, methanol, and ethanol and the like arepreferred. The solvent is evaporated and the residue furtherchromatographed using column chromatography, thin layer chromatography,preparative layer chromatography, high pressure liquid chromatographyand the like, with silica gel, aluminum oxide, ion exchange resins,dextran gels and the like, as the chromatographic medium, with varioussolvents and combinations of solvents as the eluent. Thin layer, highpressure, liquid and preparative layer chromatography may be employed todetect the presence of, and to isolate the instant compound. The use ofthe foregoing techniques as well as others known to those skilled in theart, will afford purified compositions containing the instant compound.The presence of the desired compound is determined by analyzing thevarious chromatographic fractions for biological activity againsttapeworm or against Giardia lamblia, or physico-chemicalcharacteristics. The structures of the instant compound has beendetermined by detailed analysis of the various spectral characteristicsof the compounds, in particular their nuclear magnetic resonance, mass,ultraviolet and infrared spectra. The compound has a molecular formulaof C₄₃ H₆₃ NO₁₂ and a molecular weight of 785.

In the mass spectral analysis of the instant compound no molecular ionwas observed, however, the following ions and their proposed empericalformulas are given below (M⁺ is the molecular ion).

    ______________________________________                                        Molecular Emperical                                                           Peak     Weight    Formula    Derivative                                      ______________________________________                                        M.sup.+ app                                                                            574.3879  C.sub.34 H.sub.54 O.sub.7                                                                M.sup.+ -fumaric acid                                    556.3760  C.sub.34 H.sub.52 O.sub.6                                                                M.sup.+ app-water                                        538.3654  C.sub.34 H.sub.50 O.sub.5                                                                M.sup.+ app-2 water                                      542.3563  C.sub.33 H.sub.50 O.sub.6                                                                M.sup.+ app-methanol                                     404       C.sub.24 H.sub.76 O.sub.5                                           318.2153  C.sub.20 H.sub.30 O.sub.3                                           234.1617  C.sub.15 H.sub.22 O.sub.2                                           211.0475  C.sub.9 H.sub.9 NO.sub.5                                   ______________________________________                                    

The 300 MHz proton nuclear magnetic resonance spectrum of this compound,carried out in deuterated chloroform with tetramethyl silane as aninternal standard is shown in the attached FIGURE.

In addition, a ¹³ C nuclear magnetic resonance spectrum at 75.1 MHz (15mg of the compound in 0.35 ml of CDCl₃ at 20° C.) reveals the followingchemical shifts in ppm downfield relative to tetramethyl silane as aninternal standard: 5.0, 7.0, 9.8, 10.8, 13.8, 15.3, 17.6, 20.2, 21.6,25.4, 25.9, 32.3, 34.4, 35.5, 36.8, 38.0, 39.9, 41.3, 41.7, 55.7, 70.5,71.3, 73.3, 76.3, 81.5, 82.8, 99.6, 115.3, 122.6, 125.6, 127.7, 132.9,133.7, 133.8, 134.9, 143.1, 145.2, 147.2, 164.3, 164.5, 172.6, 176.1,and 198.5.

Based on these experimental data, the instant compound is believed tohave the following structural formula: ##STR1##

The novel compound of this invention has significant parasiticidalactivity. In particular the compound has significant activity againsttapeworms. The compound is also of value as an insecticide. For example,the compound is effective against the larvae of Lucilia cuprina. Inaddition, the compound has anticoccidial and antigiardial (Giardialamblia) activity.

Giardiasis is caused by the parasite Giardia lamblia and is consideredto be the most common intestinal parasitic infection in the UnitedStates and in some other countries. The parasite is cosmopolitan and canstrike anyone regardless of their area of residence or social position.Children, however, are very often victims of the parasite. The parasitecauses a variety of intestinal symptoms, such as prolonged diarrhea,abdominal cramps, stomach pain, severe weight loss, fatigue, nausea, andflatulence. While the symptoms can be severe, the disease is generallynot fatal. Giardiasis can also cause malabsorption of nutrients and evenretarded growth. Furthermore, giardiasis can mimic the symptoms of otherconditions such as ulcers, and gall bladder attacks. If misdiagnosed, apatient may have a series of costly, needless tests and even surgery,which further delays the actual treatment of the giardiasis.

The infection is generally treated with one of three drugs: Atabrine,Flagyl or furazolidone. However each of these drugs is known to causeserious adverse side effects. The search for a safe effective treatmentof giardiasis thus is continuing. See L. K. Altman, The New York Times,pg C-1, 10 June 1980.

Trichomonas is an infection of the lower genitourinary tract, which maybe induced in men and women by the protozoan parasite Trichomonasvaginalis. The infection may produce a few symptoms of such extremediscomfort and morbidity that intervention from a gynecologist or aurologist is necessary. The disease is of cosmopolitan distribution andapparently 10-25% of sexually mature females and 25-80% of theirconsorts are involved (E. A. Steck, The Chemotherapy of ProtozoaDiaseases, Vol. II, Section 3, 17-1, 1971). Trichomoniasis is presentlytreated with Flagyl (metronidazole), which, as noted above has seriousside effects associated with its administration.

The compound is particularly active against tapeworms, a form of cestodeparasites, which infect man and animals. The tapeworm is a commonparasite which, although often symptomless, can lead to variousgastrointestinal and other disorders. Tapeworms such as Hymenolepisdiminuta, Hymenolepis nana, Taenia saginata, Taenia solium andDiphyllobothrium latum are commonly found in such infections.

When the compound of this invention is administered for the treatment oftapeworms, oral administration is preferred.

The active compound of the present invention, when used to treat theabove disorders, is orally administered, for example, with an inertdiluent, an organic solvent or with an assimilable edible carrier, orthey may be enclosed in hard or soft gelatin capsules, or they may becompressed into tablets, or they may be incorporated directly with thefood of the diet. For oral therapeutic administration, the activecompound of this invention may be incorporated with excipients and usedin the form of tablets, troches, capsules, elixirs, suppositories,suspensions, syrups, wafers, chewing gum, and the like. The amount ofactive compound in such therapeutically useful compositions orpreparations is such that a suitable dosage will be obtained.

The tablets, troches, pill, capsules and the like may also contain thefollowing: a binder such as gum tragacanth, acacia, corn starch orgelatin; an excipient such as dicalcium phosphate; a disintegratingagent such as corn starch, potato starch, alginic acid and the like; alubricant such as magnesium stearate; and a sweetening agent such assucrose, lactose or saccharin may be added or a flavoring agent such aspeppermint, oil of wintergreen, or cherry flavoring. When the dosageunit form is a capsule, it may contain in addition to materials of theabove type, a liquid carrier such as a fatty oil. Various othermaterials may be present as coatings or to otherwise modify the physicalform of the dosage unit, for instance, tablets, pills or capsules may becoated with shellac, sugar or both. A syrup or elixir may contain theactive compounds, sucrose as a sweetening agent, methyl and propylparabens as preservatives, a dye and a flavoring such as cherry ororange flavor. Of course, any material used in preparing any dosage unitform should be pharmaceutically pure and substantially non-toxic in theamount employed.

When used in the above formulation, the instant compound is employed atdosages sufficient to treat tapeworm infections. The dosages may begiven a single daily dose or in divided dosages throughout the day. Thespecific dose given to a patient will vary with the severity of thecondition, and the weight of the patient. As such, dosages of from about1 to 25 mg/kg/day have been found to be effective. Such doses may bedivided to provide for up to 4 administrations per day. The treatmentmay be carried out over several days to ensure that the parasite iscompletely destroyed. Generally, treatment period of from 1 to 21 daysare adequate.

The following examples are being provided in order that the instantinvention may be more fully understood. Such examples are not to beconstrued as being limitative of the invention.

EXAMPLE 1 Fermentation

A lyophilized tube of Streptomyces hygroscopicus MA-5285 is asepticallytransferred into 54 ml of Medium A in a 250 ml baffled Erlenmeyer flaskand incubated at 28° C. for 2 days on a rotary shaking machine at 220rpm in a 2 inch radius circular orbit. At the end of this timeapproximately 40 ml of the contents of the flask are used to inoculate300 ml of Medium F in a 2 l unbaffled Erlenmeyer flask, which is thenincubated for 4 days at 28° C. on a rotary shaking machine at 150 rpm ina 2 inch radius circular orbit.

EXAMPLE 2

The contents of a lyophilized tube of MA-5285 is transferred asepticallyto a 250 ml baffled Erlenmeyer flask containing 54 ml of Medium A. Theinoculated flask is incubated for 4 days at 28° C. on a rotary shakingmachine at a speed of 220 rpm on a 2 inch radius circular orbit. At theend of this time, 2.0 ml of this material is used to inoculate a 250 mlErlenmeyer flask containing 44 ml of either Medium B, G or H. Theseinoculated flasks are incubated at 28° C. for 4 days on a rotary shakerat a speed of 220 rpm in a 2 inch radius circular orbit.

EXAMPLE 3 Extraction and Isolation

One liter of the whole broth from Example 1 is extracted twice and oneliter of ethyl acetate; the layers are separated.

After evaporation in vacuo of the organic layer, the residue (750 mg) istaken up in 10 ml of methanol-methylene chloride (20:1) and fractionatedon a 4.3 liter Sephadex LH-20 column and eluted with methanol. Thecolumn is eluted taking 300 fractions of 20 ml each.

Tapeworm assay of the resulting fractions indicates activity to berestricted to one single zone eluted between 0.79 and 0.84 columnvolumes.

The tapeworm-active mixture is then evaporated, in vacuo, to dryness(solid residue: 65 mg), redissolved in 2 ml of methylenechloride-methanol (93:7) and further fractionated by silica gel columnchromatography using E. Merck Lobar silica gel column, 2.5 cm indiameter and 31 cm. long, eluting with a methylene chloride-methanol,gradient from 93:7 to 80:20. The column is eluted taking 300 fractionsof 6 ml each.

Tapeworm assay of the resulting fractions indicates the activity to berestricted to one zone eluted between 1.2 and 1.6 column volumes.

The active mixture thus obtained is evaporated in vacuo (solid residueweighs 2.5 mg), redissolved in 0.4 ml 75% methanol and furtherfractionated by reversed-phase HPLC (Whatman Magnum 9 ODS-3 columneluted with CH₃ CN-0.01 M, pH 3, ammonium phosphate, gradient from 40:60to 95:5).

Tapeworm assay revealed that activity is found in one zone, elutedbetween 6.5 and 7.5 column volumes (weight: 1.5 mg approximately).

Final purification of the main, active component to eliminate traceimpurities, is achieved by silica gel HPLC (Whatman 25 cm long×4 mm IDsilica gel column; chloroform-acetic acid 99:1). The pure compoundweight obtained is 1.4 mg.

EXAMPLE 4

The assay used to determine the presence of the tapeworm activefractions in Example 3 is as set forth below.

Rats are infected with mature Hymenolipsis diminuta and the infectionsare allowed to mature. The rats are then administered and compound beingtested by gavage. After from 2 to 6 days the rats are sacrificed andnecropsied. The small intestine is examined for tapeworms.

EXAMPLE 5

In each well of a multiwell plate is placed 1.4 ml of Diamond's TPSmedium (See Table I) at pH 7.05, 10% by volume of heat-inactivated fetalbovine serum, and 1% by volume of an antibiotic-antimycotic solution(see Table II). A suspension of Giardia lamblia is centrifuged at 2,500xg for 6 minutes. The pelleted cells are resuspended in a small volumeof Diamond's medium, the cells are counted and each well inoculated withapproximately 10⁶ organisms. The fractions obtained during the isolationprocedure are evaporated to dryness and the residue dissolved indimethylsulfoxide. The wells are then inoculated with various fractionsof the compound to determine its presence. The plates are incubated for24 hours at 37° C. in an anaerobic Gas Pak jar. After 24 hours ofincubation, each well is mixed and counted for viable organisms using ahemacytometer. The percentage of survival is determined by comparing thetreated wells to controls treated with dimethylsulfoxide. The presenceof the compound in a particular fraction is noted when the well analysisindicates that a substantial portion of the Giardia have been killed.

                  TABLE I                                                         ______________________________________                                        Composition of Diamond's TPS Medium                                           Ingredients            Amounts                                                ______________________________________                                        Trypticase (BBL)       1.00 g.                                                Panmede, liver digest P&B                                                                            2.00 g.                                                Glucose                0.50 g.                                                L-cysteine monohydrochloride                                                                         0.10 g.                                                Ascorbic acid          0.02 g.                                                Sodium chloride        0.50 g.                                                Potassium phosphate. monobasic                                                                       0.06 g.                                                Potassium phosphate dibasic, anhydrous                                                               0.10 g.                                                Water, glass distilled 87.50 ml.                                                pH adjusted to 7.0 with 1N NaOH                                             ______________________________________                                    

                  TABLE II                                                        ______________________________________                                        Antibiotic Antimycotic Solution (100X)                                        Ingredients            Amounts                                                ______________________________________                                        Penicillin             10,000 units                                           Streptomycin           10,000 mcg                                             Fungizone®            25 mcg                                              ______________________________________                                         Prepared in normal saline                                                

What is claimed is:
 1. A compound having the formula: ##STR2##
 2. Acomposition useful for the prevention and treatment of parasiticinfections which comprises an inert carrier having the compound of claim1 incorporated therein.
 3. The composition of claim 2 wherein theparasitic infection is a tapeworm.
 4. A method for the prevention andtreatment of parasitic infections which comprises administering to ananimal infected with parasites, an effective amount of the compound ofclaim
 1. 5. The composition of claim 4 wherein the parasitic infectionis a tapeworm.